Flow cytometers are commonly employed in the medical industry to analyze particles in a patient's body fluid (e.g., blood cells) as an adjunct to the diagnosis and treatment of disease. As a non-limiting example, in the course of chemotherapy treatment, such instruments may be used to sort and collect healthy blood cells (stencils) from a quantity of blood that has been removed from a patient's bone marrow prior to chemotherapy. Once a chemotherapy treatment session is completed, a collected quantity of these cells is then reinjected back into the patient, to facilitate migration and healthy blood cell reproduction.
For this purpose, as diagrammatically illustrated in FIG. 1, particles 10 to be analyzed, such as cells of a centrifuged blood sample stored in a container 12, are analyzed by injecting the particles into a (pressurized) continuous or uninterrupted carrier fluid (e.g., saline) 14, and directing the carrier fluid along a channel 15 that passes through the output beam 16 emitted by an optical illumination subsystem, such as one or more lasers 18. Located optically in the path of the laser output beam 16 after its being intercepted by the carrier fluid passing therethrough are one or more photodetectors of a photodetector subsystem 21.
This photodetector subsystem 71 is positioned to receive light as modulated by the contents of (particles/cells within) the fluid in the channel 15, including that reflected off a cell, the blocking of light by a cell, and a light emission from a fluorescent dye antibody attached to a cell. In order to avoid confusion as to which photodetector output signal is representative of which illuminated cell, the fluid flow channel through the cytometer is configured and sized to pass the particles or cells only one cell at the time through an intersection location 19 with the laser's output beam 16. As a consequence, as the output signals from the photodetector of interest is irradiated by the laser beam to the time when a droplet containing the cell of interest eventually separates from the carrier fluid stream.
The individual droplets 23 produced by the droplet generator 25 do not form immediately at the droplet generator's exit port 27, but rather break off naturally and in random fashion at location 29 downstream of the exit port 27. The point in space 29 downstream of the exit port 27 at which the droplets individually form may be adjusted by varying the parameters of the drive signal to the piezoelectric transducer of the droplet generator 25, and may be defined so as to cause the droplets 23 to become synchronized with the frequency of the piezo vibration of the droplet generator 25. As a non-limiting example, the acoustic drive frequency applied to the droplet generator 25 may be on the order for from four to one hundred Khz, at a fluid pressure on the order of from three to seventy psi.
Once the droplets 23 have been individually formed in a spaced apart sequence, they may be controllably sorted by means of droplet sorter 30 into a collection container 41, or allowed to pass unsorted along a main travel path 24 into an aborted or discarded waste container 43. The droplet sorter's electrostatic charging collar 31 may comprise a metallic ring surrounding that point in the droplet stream 22 where the individual droplets 23 separate from the fluid stream, and is typically several droplets in length. It is positioned vertically downstream of the exit port 27 of the droplet generator 25, and upstream of an subsystem 21 are modulated by the particles in the carrier fluid, each modulation signal can be associated with a respective cell in the fluid carrier stream.
If the output of the photodetector subsystem 21 satisfies prescribed `sort` criteria associated with one or more parameters of a desired cell, then subsequent to a `sort delay`, the photodetector output signal may be used to control the sorting of a droplet of the carrier fluid containing that cell (by means of a downstream droplet sorter 30), once that droplet is formed by into a stream of droplets 23 by means of an acoustically (e.g., piezoelectric) driven droplet generator 25. For this purpose, the photodetector output is typically digitized and then analyzed by a cell type mapping or identification algorithm executed by an associated supervisory control processor. Based upon this analysis, the control processor instructs the droplet sorter to sort or abort the droplet.
The sort delay is the period of time that elapses between the instant in time at which the photodetector generates an output signal for that cell as it is illuminated by the laser beam at intersection location 19 and the time at which that portion of the fluid stream containing that cell breaks off into a droplet at some downstream location 29. The sort delay may be defined means of by a delay timer, such as a counter driven by a high speed sort control clock, so that the output of the counter indicates the time difference between the time that the portion of region of the carrier fluid containing the cell associated set of electrostatic (opposite polarity, high voltage) deflection plates 33 and 35 between which the stream of charged droplets 23 pass as they travel downwardly towards the collection and waste containers.
Under the control of a cell analysis and sorting routine executed by the system workstation 50, a prescribed charging voltage is selectively applied via deflection control circuitry 52 to the charging collar 31 at a time determined by the sort delay, thereby charging that segment of the cell-containing fluid stream, so that any droplet breaking off from the stream at that point and containing the cell of interest will carry the charge induced by the collar. Then, as an individual charged droplet carrying this charge (one of which is shown at 23C) passes between the two opposite polarity high voltage deflection plates 33 and 35, it is attracted to the plate with the opposite charge, while being simultaneously repelled by the plate with the same or like charge. For a droplet containing a cell to be sorted, this electrostatic steering action directs the charged droplet 23C along a path 26 to one side of the travel path 24 of the main stream, and into the collection container 41 placed on the side of the path of the main stream of droplets.
The location of the point 29 where an individual droplet 23 forms or breaks off from the continuous fluid stream 22 is critical to accurate sorting of the droplets, since only a droplet that breaks off from the stream at the time of the applied sort charge will be deflected by the deflection plates, and subsequently collected in the target sorting container. As described above, for any given cell 10 within the fluid stream 22, there is a `sort` delay between the time at which the photodetector subsystem 21 generates an output signal for that cell and the time at which a droplet 23 containing that cell breaks off from the fluid stream. As described previously, during this sort delay, the output signal from the photodetector is digitized and then analyzed-processed by a droplet sort routine executed by the system's control processor (controlled by or installed within workstation 50) to determine whether the droplet containing the cell of interest is to be sorted into the collection container 41 or aborted into the waste container 43.
Since sort delay is affected, inter alia, by the pressure of the carrier fluid, size and surface characteristics of the droplet generator exit port, the viscosity of the carrier fluid, and the amplitude of the piezo vibration, a preliminary calibration cycle is conducted to accurately locate the droplet formation point. As a non-limiting example, this may be accomplished by manually placing the droplet formation point 29 at a predetermined distance from the laser intersection point 19, using a microscope objective, or a video system, to observe the fluid steam 22. By strobing a light emitting diode in sync with the excitation frequency of the drive signal to the droplet generator 25, the droplets 23 formed from the fluid stream 22 will appear to be stationary. By increasing or decreasing the amplitude of the drive signal to a piezoelectric actuator 28, the droplet formation point 29 can be moved closer or farther away from the laser intersection point 19. The system operator can then adjust the point at which the droplets first form to a reference or positioning mark.
Next, the operator inputs to the sorting system a sort delay time that has been determined on the basis of previous experimentation, so as to place the system within several droplets of the actual sort delay time. In order to bring the system to within one droplet of accuracy, the operator sets up and runs a calibration sort operation, using test beads, which mimic biological cells in terms of size. The beads are sorted onto a slide, and the slide is observed with a microscope in order to determine if the number of beads on the slide coincides with the number of beads the system reported as having sorted. If the numbers do not coincide, then the system is adjusted by changing the sort delay time, or by moving the droplet formation point by adjusting the acoustic drive signal. This operation is iteratively repeated as necessary until the beads counts are correct. With the system thus initially calibrated, it may then be monitored visually for drift, with the operator observing the fluid stream and droplets for movement. To verify that the sort parameters remain the same, the slide and bead analysis sequence described above may be repeated.
Because the particle (e.g., blood cell) processing rate of a flow cytometer is often limited to a relatively slow data rate (e.g., a range on the order of from ten to thirty thousand cells per second), then for commonplace yield rates of desired cells on the order of only five percent or less, the time required to collect or harvest a highly purified quantity of cells (e.g., on the order of a million or more), may be as long as six hours. One of the reasons for this relatively lengthy harvesting time is the fact that the sorting routine executed by the system's supervisory computer will customarily cause droplets that have been determined to contain (have in close proximity) `events` other than only good cells to be aborted (discarded) into the waste container, even though such droplets may also contain desired cells. Once such aborted droplets containing good cells are discarded they cannot be reclaimed.
Unfortunately harvesting time cannot be reduced by simply increasing the data rate (droplet processing speed) through the cytometer, since increasing the data rate effectively decreases the separation cells and droplets, and thereby places undesired components or anomalies spatially closer to the desired cells, causing more cells (up to fifty percent) to be aborted. Also, increasing the data rate can increase system jitter. Either of these conditions may place an anomaly sufficiently close to the type of cell of interest as to cause the droplet containing the desired cell to be aborted.